The bulk physical properties of biological membranes are governed by the composition of their phospholipid components and consequently membrane phospholipid structure in Escherichia coli, as in other organisms, is rigorously controlled. The long-term goal of this research program is to define the physiological processes that determine and modify membrane phospholipid structure. The glycerol phosphate acyltransferase system specifies the initial phospholipid fatty acid composition, but our results show what a metabolic cycle also exists for the removal and replacement of 1-position fatty acids in phosphatidylethanolamine (PtdEtn). The modification of proteins with fatty acids is and important post-translational determinant of a protein's subcellular distribution and function and we have shown that PtdEtn turnover is related to the utilization of 1-position acyl moieties for amino terminal acylation of the major outer membrane lipoprotein. The resulting 2-acylglycerophosphoethanolamine (2-acyl-GPE) is then recycled into PtdEtn by a 1-position-specific acyl carrier protein (ACP)-dependent acyltransfersse. Our data indicate that a single polypeptide catalyzes both 2-acyl-GPE acyltransferase and acyl-ACP synthetase. This enzyme system also provides a mechanism for the assimilation of extracellular fatty acids into PtdEtn that circumvents a requirement for prior conversion to an acyl-coenzyme A intermediate. These findings represent important conceptual advances in that we have defined new metabolic pathways related to fatty acid turnover in membrane phospholipids, lipoprotein acylation and incorporation of exogenous fatty acids into phospholipid. The existence of these pathways was deduced from in vivo labeling experiments and the goal of this grant proposal is to corroborate and refine our hypotheses by focusing on the biochemistry and genetics of the enzymes that catalyze these metabolic conversions. The specific aims are: (1) to determine the catalytic mechanism and physical properties of purified 2-acyl- GPE acyltransferase/acyl-ACP synthetase and to isolate mutants defective in this enzyme (2) to develop in vitro assays for phospholipid:lipoprotein transacylases and to biochemically characterize these enzymes; and (3) to define the mechanism for fatty acid uptake and isolate mutants defective in the transport machinery.